Identification using Mass Spectrometry

Mass Spectrometry detection can provide mass spectra, isotope patterns, and/or signals for selected ions. Although mass spectra can be highly specific for an analyte, match values differ depending on the particular software used which makes it impossible to set generic guidance on match values for identification. This means that laboratories that use spectral matching for identification need to set their own criteria and demonstrate that these are fit- forpurpose. Guidance for identification based on MS spectra is limited to some recommendations whereas for identification based on selected ions more detailed criteria are provided.

Reference spectra for the analyte should be generated using the same instruments and conditions used for analysis of the samples. If major differences are evident between a published spectrum and the spectrum generated within the laboratory, the latter must be shown to be valid. To avoid distortion of ion ratios the concentration of the analyte ions must not overload

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the detector. The reference spectrum in the instrument software can originate from a previous injection (without matrix present), but is preferably obtained from the same analytical batch.

In case of full scan measurement, careful subtraction of background spectra, either manual or automatic, by deconvolution or other algorithms, may be required to ensure that the resultant spectrum from the chromatographic peak is representative. Whenever background correction is used, this must be applied uniformly throughout the batch and should be clearly recorded.

Identification relies on the correct selection of ions. They must be sufficiently selective for the analyte in the matrix being analysed and in the relevant concentration range. Molecular ions, (de)protonated molecules or adduct ions are highly characteristic for the analyte and should be included in the measurement and identification procedure whenever possible. In general, and especially in single-stage MS, high m/z ions are more selective than low m/z ions (e.g. m/z < 100). However, high mass m/z ions arising from loss of water or loss of common moieties may be of little use. Although characteristic isotopic ions, especially Cl or Br clusters, may be particularly useful, the selected ions should not exclusively originate from the same part of the analyte molecule. The choice of ions for identification may change depending on background interferences. In HRMS, the selectivity of an ion of the analyte is determined by the narrowness of the mass extraction window (MEW) that is used to obtain the extracted ion chromatogram (EIC). The narrower the MEW, the higher the selectivity. However, the minimum MEW that can be used relates to mass resolution.

EICs of sample extracts should have peaks of similar retention time, peak shape and response ratio to those obtained from calibration standards analysed at comparable concentrations in the same batch. Chromatographic peaks from different selective ions for the analyte must fully overlap

Different types and modes of mass spectrometric detectors provide different degrees of selectivity , which relates to the confidence in identification. The requirements for identification are given in the table 3.1.

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The relative intensities or ratios of selective ions, expressed as a ratio relative to the most intense ion, that are used for identification, should match with the reference ion ratio. The reference ion ratio is the average obtained from solvent standards measured in the same sequence and under the same conditions as the samples. Standards in matrix may be used instead of solvent standards as long as they have been demonstrated to be free of interferences for the ions used at the retention time of the analyte. For determination of the reference ion ratio, responses outside the linear range should be excluded.

As long as sufficient sensitivity and selectivity are obtained for both ions, and responses are within the linear range, ion ratios in unit mass resolution MS/MS have shown to be consistent and should not deviate more than 30%

(relative) from the reference value.

For accurate mass measurement / high resolution mass spectrometry, the variability of ion ratios is not only affected by S/N of the peaks in the extracted ion chromatograms, but may also be affected by the way fragment ions are generated, and by matrix. For example, the range of precursor ions selected in a fragmentation scan event ('all ions', precursor ion range of 100 Da, 10 Da, or 1 Da) results in different populations of matrix ions in the collision cell which can affect fragmentation compared to solvent standards. Furthermore, the ratio of two ions generated in the same fragmentation scan event tends to yield more consistent ion ratios than the ratio of a precursor from a full scan event and a fragment ion from a fragmentation scan event. For this reason, no generic guidance value for ion ratio can be given. Due to the added value of accurate mass measurement, matching ion ratios are less critical, however, they should be used as indicative.

Table 1.4 summarizes mass spectrometry identification requirements.

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Table 1.4 : Identification requirements for different MS techniques, SANTE11813/2017

MS detector/Characteristics

Acquisition

Requirements for identification

Resolution

Typical systems (examples)

minimum number of

ions

other

Unit mass resolution (LRMS)

Single MS quadrupole, ion

trap, TOF

full scan, limited

m/z range, SIM 3 ions

S/N ≥ 3^d Analyte peaks

from both product ions in the extracted ion

chromatograms must fully overlap. Ion

ratio from sample extracts should be within

±30% (relative) of average of

calibration standards from same sequence MS/MS triple

quadrupole, ion trap, Q-trap, Q- TOF, Q-Orbitrap

selected or multiple reaction

monitoring (SRM, MRM), mass resolution for precursor-ion

isolation equal to or better than

unit mass resolution

2 product ions

Accurate mass measurement

(HRMS)

High resolution MS: (Q-)TOF (Q-)Orbitrap FT-

ICR-MS sector MS

full scan, limited m/z range, SIM, fragmentation with or without precursor-ion

selection, or combinations

thereof

2 ions with mass accuracy ≤ 5

ppm^{a, b, c}

S/N ≥ 3^d Analyte peaks from precursor and/or product ion(s) in the extracted ion chromatograms

must fully overlap.

a: preferably including the molecular ion, (de)protonated molecule or adduct ion b: including at least one fragment ion

c: < 1 mDa for m/z < 200

d: in case noise is absent, a signal should be present in at least 5 subsequent scans

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